Ninjurin1 is known as an adhesion molecule promoting leukocyte trafficking under inflammatory conditions. However, the posttranslational modifications of Ninjurin1 are poorly understood. Herein, we defined the proteolytic cleavage of Ninjurin1 and its functions. HEK293T cells overexpressing the C- or N-terminus tagging mouse Ninjurin1 plasmid produced additional cleaved forms of Ninjurin1 in the lysates or conditioned media (CM). Two custom-made anti-Ninjurin1 antibodies, Ab1–15 or Ab139–152, specific to the N- or C-terminal regions of Ninjurin1 revealed the presence of its shedding fragments in the mouse liver and kidney lysates. Furthermore, Matrix Metalloproteinase (MMP) 9 was responsible for Ninjurin1 cleavage between Leu56 and Leu57. Interestingly, the soluble N-terminal Ninjurin1 fragment has structural similarity with well-known chemokines. Indeed, the CM from HEK293T cells overexpressing the GFP-mNinj1 plasmid was able to attract Raw264.7 cells in trans-well assay. Collectively, we suggest that the N-terminal ectodomain of mouse Ninjurin1, which may act as a chemoattractant, is cleaved by MMP9.